This work was supported by the National Natural Science Foundation of China (No. 31870746), Shenzhen Basic Research Grants (JCYJ20200109140414636), and Natural Science Foundation of Guangdong Province, China (No. 2021A1515010796) to W. L. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

1. Purification of GST-tagged TRP and His-tagged NORPA fragments
<ol>
	<li>Purify GST-tagged TRP 1261-1275 fragment
	<ol>
		<li>Transform the pGEX 4T-1 TRP 1261-1275 plasmid10 into Escherichia coli (E. coli) BL21 (DE3) cells using the CaCl2 heat-shock transformation method21. Inoculate a single colony in 10 mL of Luria Bertani (LB) medium and grow overnight at 37 &#176;C. Then, amplify the 10 mL of seeding culture in 1 L of LB medium a...

<ol>
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G., Hawley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A laboratory handbook. Second Edition. 80 (2), (2005).</li><li>Nicolas, G., Sillans, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+and+latent+effects+of+carbon+dioxide+on+insects.">Immediate and latent effects of carbon dioxide on insects.</a> Annual Review of Entomology. 34 (1), 97-116 (1989).</li><li>Arachea, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent+selection+for+enhanced+extraction+of+membrane+proteins.">Detergent selection for enhanced extraction of membrane proteins.</a> Protein Expression and Purification. 86 (1), 12-20 (2012).</li><li>Hellmich, U. 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INAD, which contains five PDZ domains, is the core organizer of Drosophila phototransduction machinery. Previous studies showed that INAD PDZ3 binds to the TRP channel C-terminal tail with exquisite specificity (KD = 0.3 &#181;M)18. INAD PDZ45 tandem interacts with NORPA 863-1095 fragment with an extremely high binding affinity (KD = 30 nM). These findings provide a solid biochemical basis to design the affinity purification plus competition strategy, which enables t...

Phototransduction is a process where absorbed photons are converted into electrical codes of neurons. It exclusively relays opsins and the following G protein-coupled signaling cascade in both vertebrates and invertebrates. In Drosophila, by using its five PDZ domains, scaffold protein inactivation-no-after-potential D (INAD) organizes a supramolecular signaling complex, which consists of a transient receptor potential (TRP) channel, phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA), and eye-specific protein kinase C (ePKC)1. The formation of this supramolecular signaling complex guarantees the correct subcellular localization, high efficiency, and specificity of Drosophila phototransduction machinery. In this complex, light-sensitive TRP channels act as downstream effectors of NORPA and mediate calcium influx and the depolarization of photoreceptors. Previous studies showed that the opening of the Drosophila TRP channel is mediated by protons, disruption of the local lipid environment, or mechanical force2,3,4. The Drosophila TRP channel also interacts with calmodulin5 and is modulated by calcium by both positive and negative feedback6,7,8.
So far, electrophysiology studies on the gating mechanism of Drosophila TRP and TRP-like (TRPL) channels were based on excised membrane patches, whole-cell recordings from dissociated wild-type Drosophila photoreceptors, and hetero-expressed channels in S2, SF9, or HEK cells2,9,10,11,12,13, but not on purified channels. The structural information of the full-length Drosophila TRP channel also remains unclear. In order to study the electrophysiological properties of purified protein in a reconstituted membrane environment and to gain structural information of the full-length Drosophila TRP channel, obtaining purified full-length TRP channels is the necessary first step, similar to the methodologies used in mammalian TRP channel studies14,15,16,17.
Recently, based on the assembling mechanism of INAD protein complex18,19,20, an affinity purification plus competition strategy was first developed to purify the TRP channel from Drosophila head homogenates by streptavidin beads5. Considering the low capacity and expensive cost of streptavidin beads, an improved purification protocol is introduced here that uses His-tagged bait protein and corresponding low-cost Ni-beads with much higher capacity. The proposed method will help to study the gating mechanism of the TRP channel from structural angles and to measure the electrophysiological properties of the TRP channel with purified proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacterial strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3) Competent Cells</td>
			<td>Novagen</td>
			<td>69450</td>
			<td>Protein overexpression</td>
		</tr>
		<tr>
			<td>Experiment models</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D.melanogaster: W1118 strain</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>BDSC:3605</td>
			<td>Drosophila head preparation</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20/30/40 mesh stainless steel sieves</td>
			<td>Jiufeng metal mesh company</td>
			<td>GB/T6003.1</td>
			<td>Drosophila head preparation</td>
		</tr>
		<tr>
			<td>30% Acrylamide-N,N&#8242;-Methylenebisacrylamide(29:1)</td>
			<td>Lablead</td>
			<td>A3291</td>
			<td>SDS-PAGE gel preparation</td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Invitrogen</td>
			<td>HC2005</td>
			<td>SDS-PAGE gel preparation</td>
		</tr>
		<tr>
			<td>Cocktail protease inhibitor</td>
			<td>Roche</td>
			<td>05892953001</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Coomassie brilliant blue R-250</td>
			<td>Sangon Biotech</td>
			<td>A100472-0025</td>
			<td>SDS-PAGE gel staining</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol (DTT)</td>
			<td>Sangon Biotech</td>
			<td>A620058-0100</td>
			<td>Size-exclusion column buffer preparation</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt (EDTA)</td>
			<td>Sangon Biotech</td>
			<td>A500838-0500</td>
			<td>Size-exclusion column buffer preparation</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sangon Biotech</td>
			<td>A610235-0005</td>
			<td>SDS-PAGE buffer preparation</td>
		</tr>
		<tr>
			<td>Glutathione Sepharose 4 Fast Flow beads</td>
			<td>Cytiva</td>
			<td>17513202</td>
			<td>Affinity chromatography</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sangon Biotech</td>
			<td>A500529-0001</td>
			<td>Elution buffer preparation for Ni-column</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td>Sangon Biotech</td>
			<td>A600168-0025</td>
			<td>Induction of protein overexpression</td>
		</tr>
		<tr>
			<td>LB Broth Powder</td>
			<td>Sangon Biotech</td>
			<td>A507002-0250</td>
			<td>E.coli. cell culture</td>
		</tr>
		<tr>
			<td>L-Glutathione reduced (GSH)</td>
			<td>Sigma-aldrich</td>
			<td>G4251-100G</td>
			<td>Elution buffer preparation for Glutathione beads</td>
		</tr>
		<tr>
			<td>Ni-Sepharose excel beads</td>
			<td>Cytiva</td>
			<td>17371202</td>
			<td>Affinity chromatography</td>
		</tr>
		<tr>
			<td>N-Dodecyl beta-D-maltoside (DDM)</td>
			<td>Sangon Biotech</td>
			<td>A610424-001</td>
			<td>Detergent for protein purification</td>
		</tr>
		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-aldrich</td>
			<td>T9281-100ML</td>
			<td>SDS-PAGE gel preparation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sangon Biotech</td>
			<td>E607008-0500</td>
			<td>Homogenization buffer for E.coli. cell</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Lablead</td>
			<td>P0754-25G</td>
			<td>Protease inhibitor</td>
		</tr>
		<tr>
			<td>Prestained protein marker</td>
			<td>Thermo Scientific</td>
			<td>26619/26616</td>
			<td>Prestained protein ladder</td>
		</tr>
		<tr>
			<td>Size exclusion column (preparation grade)</td>
			<td>Cytiva</td>
			<td>28989336</td>
			<td>HiLoad 26/60 Superdex 200 PG column</td>
		</tr>
		<tr>
			<td>Size exclusion column (analytical grade)</td>
			<td>Cytiva</td>
			<td>29091596</td>
			<td>Superose 6 Increase 10/300 GL column</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sangon Biotech</td>
			<td>A501218-0001</td>
			<td>Protein purification buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sangon Biotech</td>
			<td>A500228-0001</td>
			<td>SDS-PAGE gel/buffer preparation</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-aldrich</td>
			<td>T1503-10KG</td>
			<td>Protein purification buffer preparation</td>
		</tr>
		<tr>
			<td>Ultrafiltration spin column</td>
			<td>Millipore</td>
			<td>UFC901096/801096</td>
			<td>Protein concentration</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>DENVER</td>
			<td>APX-60</td>
			<td>Metage of Drosophila head</td>
		</tr>
		<tr>
			<td>Desk-top high-speed refrigerated centrifuge for 15mL and 50mL conical centrifugation tubes</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Protein concentration</td>
		</tr>
		<tr>
			<td>Desk-top high-speed refrigerated centrifuge 1.5mL centrifugation tubes</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td>Centrifugation of Drosophila head lysate after homogenization</td>
		</tr>
		<tr>
			<td>Empty gravity flow column (Inner Diameter=1.0cm)</td>
			<td>Bio-Rad</td>
			<td>738-0015</td>
			<td>TRP protein purification</td>
		</tr>
		<tr>
			<td>Empty gravity flow column (Inner Diameter=2.5cm)</td>
			<td>Bio-Rad</td>
			<td>738-0017</td>
			<td>Bait and competitor protein purification from E.coli.</td>
		</tr>
		<tr>
			<td>Gel Documentation System</td>
			<td>Bio-Rad</td>
			<td>Universal Hood II Gel Doc XR System</td>
			<td>SDS-PAGE imaging</td>
		</tr>
		<tr>
			<td>High-speed refrigerated centrifuge</td>
			<td>Beckman coulter</td>
			<td>Avanti J-26 XP</td>
			<td>Centrifugation of E.coli. cells/cell lysate</td>
		</tr>
		<tr>
			<td>High pressure homogenizer</td>
			<td>UNION-BIOTECH</td>
			<td>UH-05</td>
			<td>Homogenization of E.coli. cells</td>
		</tr>
		<tr>
			<td>Liquid nitrogen tank</td>
			<td>Taylor-Wharton</td>
			<td>CX-100</td>
			<td>Drosophila head preparation</td>
		</tr>
		<tr>
			<td>Protein purification system</td>
			<td>Cytiva</td>
			<td>AKTA purifier</td>
			<td>Protein purification</td>
		</tr>
		<tr>
			<td>Refrigerator (-80&#176;C)</td>
			<td>Thermo</td>
			<td>900GP</td>
			<td>Drosophila head preparation</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>MAPADA</td>
			<td>UV-1200</td>
			<td>OD600 measurement of E.coli. cells</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop 2000c</td>
			<td>Determination of protein concentration</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman coulter</td>
			<td>Optima XPN-100 Ultracentrifuge</td>
			<td>Ultracentrifugation</td>
		</tr>
	</tbody>
</table>

purification of endogenous drosophila transient receptor potential channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

In this article, a protein purification method is demonstrated to purify endogenous Drosophila TRP channel (Figure 1).
First, recombinant protein expression and purification are applied to obtain the bait and competitor proteins. Then, a GST-tagged TRP 1261-1275 fragment is expressed in E. coli BL21 (DE3) cells in LB medium and purified using glutathione beads and a size-exclusion column (Figure 2). The samples were ...

Watch this Scientific Journal Video about Purification of Endogenous Drosophila Transient Receptor Potential Channels at JoVE.com

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...

In this protocol, we demonstrate the purification process to obtain purified, full-length TRP channels, which is the necessary first step to study electrophysiological properties, and gain structural information of the full-length Drosophila TRP channel. Based on the sampling mechanism of the INAD protein complex and using low cost nickel beads with much higher capacity, sufficient amount of TRP channels can be purified from fly heads. Demonstrating the procedure will be Yuyang Liu, a technician from our lab.To begin, transform the pGEX 4T-1 TRP 1261-1265 plasmid into E.coli BL21 cells, using the calcium chloride heat shock transformation method. Inoculate a single colony in 10 milliliters of Luria Bertani medium, and grow overnight at 37 degrees Celsius. Add the 10 milliliters of seeding culture into one liter of Luria Bertani medium and incubate at 37 degrees Celsius.After the optical density of the cells reaches 0.5, cool the cells to 16 degree Celsius, and add 0.1 millimolar isopropyl-beta-D-1-thiogalactopyranoside. After overexpression, pellet one liter of cultured cells by centrifugation, and resuspend in 40 milliliters of phosphate-buffered saline. Load the resuspended cells into a high pressure homogenizer, pre-cooled to four degrees Celsius.Slowly increase the homogenizer pressure to 800 bar. Open the inlet tab and let the resuspended cells circularly pass through a valve with narrow slits. Load five milliliters of glutathione beads into a gravity flow column, and wash the beads with 50 milliliters of phosphate-buffered saline buffer, for a total of three times.Centrifuge the cell lysate from the high pressure homogenizer at 48, 384 times G.Add around 40 milliliters of the supernatant of the centrifuged cell lysate to the equilibrated glutathione beads in the gravity flow column, and incubate for 30 minutes at 4 degrees Celsius. Resuspend the glutathione beads every 10 minutes. After 30 minutes of incubation, open the column outlet tab to separate the beads and flow-through fraction.Discard the flow-through fraction, and rinse the remaining glutathione beads twice, with 50 milliliters of phosphate-buffered saline buffer. Add 15 milliliters of elution buffer to the glutathione beads, and resuspend the beads every 10 minutes. Elute the GST-tagged TRP 1261-1275 fragment into a 50 mL conical tube, and load it into a size exclusion column.Collect five milliliters of the eluted proteins per tube, and concentrate the purified fragments to one milliliter by centrifugation in an ultrafiltration spin column in a refrigerated centrifuge. To purify the His-tagged NORPA 863-1095 fragment, follow the previously demonstrated procedure using nickel beads. Collect adult flies in 50 milliliter conical centrifuge tubes, and freeze them in liquid nitrogen for 10 minutes.Vigorously shake the frozen tubes by hand and transfer the mixture to three, sequentially stacked, pre-cooled stainless-steel sieves. Shake the sieves, and sweep the fly heads off the 40 mesh sieve using a brush. Transfer them into five milliliter conical tubes and store them at minus 80 degrees Celsius.Homogenize 0.5 grams of heads in liquid nitrogen using a pre-cooled mortar and pestle. Dissolve the homogenized heads in five milliliters of 10X lysis buffer, followed by centrifugation at 20, 817 times G at four degrees Celsius for 20 minutes. The spin down supernatant is further centrifuged 100, 000 times G at four degrees Celsius for 60 minutes.Add one milliliter of nickel beads into the gravity flow column, and wash the beads three times with 10 milliliters of double distilled water at four degrees Celsius. Equilibrate the beads with 10 column volumes of lysis buffer, three times at four degrees Celsius. Add 500 microliters of 600 micromolar purified His-tagged NORPA 863-1095 protein into the nickel column, and incubate the column.Resuspend the beads every 10 minutes. Open the column outlet tap to separate the beads and flow-through fraction, and wash the nickel-beads with 10 milliliters of cold lysis buffer. Then, add cold supernatant of Drosophila head homogenate into the nickel column.After incubation, wash the beads with 10 milliliters of lysis buffer and add 500 microliters of 600 micromolar GST-tagged TRP 1261-1275 protein into the beads. Collect the eluted fraction from the gravity column. Wash the nickel beads with 10 milliliters of binding buffer.Then, add elution buffer and collect the elution fraction from the gravity flow column. Concentrate the fractions eluted from Drosophila TRP channel purification, using a four milliliter ultrafiltration spin column. Inject the sample into the size exclusion column, and elute the proteins with a reasonable flow rate.GST-tagged TRP 1261-1275 fragment was expressed in E.coli cells and purified using glutathione beads and a size exclusion column. Similarly, the His-tagged NORPA 863-1095 fragment was expressed and purified using nickel beads and size exclusion column. The eluted TRP channel is separated from the excessive GST-tagged TRP 1261-1275 peptide, by size exclusion chromatography.As a byproduct, the INAD, ePKC, NORPA 863-1095 complexes are obtained after TRP 1261-1275 peptide competition. The most important thing to remember when attempting this protocol, is homogenize the fly heads in liquid nitrogen and resolve the homogenate in buffer containing a detergent called DDM. Following this procedure, we can also purify the whole INAD protein complex which can be used to study its assembly and regulation mechanisms.A potential future application of this method will be to study the structural information of the endogenous Drosophila TRP channel using Cryo-EM techniques. In addition, measuring the electrophysiological properties of purified endogenous Drosophila TRP channels in artificial bilayer lipid membrane is also feasible.

purification-endogenous-drosophila-transient-receptor-potential

Research

JoVE Journal

Biochemistry

1.6K ビュー.  Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center. このプロトコールでは、電気生理学的特性を研究するために必要な最初のステップである精製された全長TRPチャネルを取得し、全長ショウジョウバエTRPチャネルの構造情報を得るための精製プロセスを実証します。INADタンパク質複合体のサンプリング機構に基づき、はるかに高い容量を有する低コストのニッケルビーズを使用して、十分な量のTRPチャネルをフライヘッ?...

ビデオ: Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP...

a fluorescence-based assay of membrane potential for high-throughput functional study of two endogenous ion channels in two epithelial cell lines

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

expression and purification of mammalian bestrophin ion channels

Expression and Purification of Mammalian Bestrophin Ion Channels

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl-...

establishing a mouse model of a pure small fiber neuropathy with the ultrapotent agonist of transient receptor potential vanilloid type 1

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic...

examining monosynaptic connections in drosophila using tetrodotoxin resistant sodium channels

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

This article features a method to test the monosynaptic connections between neurons by employing tetrodotoxin and the tetrodotoxin-resistant sodium channel,...

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

one-step crispr-based strategy for endogenous gene tagging in drosophila melanogaster

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in...

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

visualizing and tracking endogenous mrnas in live drosophila melanogaster egg chambers

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

purification of transcripts and metabolites from drosophila heads

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic"...

Purification of Transcripts and Metabolites from Drosophila Heads

dna tension probes to map the transient piconewton receptor forces by immune cells

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over...

using linear agarose channels to study drosophila larval crawling behavior

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

isolation and purification of kinesin from drosophila embryos

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP...

Isolation and Purification of Kinesin from Drosophila Embryos

purification of low-abundant cells in the drosophila visual system

Purification of Low-abundant Cells in the Drosophila Visual System

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting...

whole mount immunolabeling of olfactory receptor neurons in the drosophila antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

isolation and purification of drosophila peripheral neurons by magnetic bead sorting

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

isolation of adeno-associated viral vectors through a single-step and semi-automated heparin affinity chromatography protocol

Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical...

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

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identifying protein-protein interaction in drosophila adult heads by tandem affinity purification (tap)

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

trap-rc, translating ribosome affinity purification from rare cell populations of drosophila embryos

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

immunofluorescence analysis of endogenous and exogenous centromere-kinetochore proteins

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)&#8212;two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 &#177; 3.6 &#181;M and a Ki of 23.2 &#177; 3.2 &#181;M. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors.

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...